P-069: EFFECTS OF QUERCETIN IN TRANSCRIPTIONAL REGULATION OF FETAL HEMOGLOBIN
Authors: CANTEIRO B., MENDES M., DELGADINHO M., OLIVEIRA K., GINETE C., GOMES M., BRITO M., GOMES A., RIBEIRO E.
Journal: HemaSphere (2022)

Purpose: Sickle cell disease (SCD) is a genetic blood disorder that affects the shape and transport of red blood cells (RBCs) in blood vessels, leading to various clinical complications. The pharmacological reactivation of Fetal Hemoglobin (HbF) is considered to be a viable therapeutic method in SCD. In this regard, hydroxyurea (HU), a powerful ribonucleotide reductase inhibitor, is being employed as a HbF-inducing pharmaceutical. However, its cytotoxicity, carcinogenic potential and variable effects limit its use. Thus, a major challenge today is to identify new agents, with high HbF-inducing activity, low cytotoxicity, and available in low- and middle-income countries, such as natural compounds. Quercetin, a natural flavonoid, has been identified as a potential HbF inducer. The main aim of this work was to evaluate Quercetin role in the reactivation of fetal hemoglobin (HbF) by analyzing the expression of globin and HbF regulatory/silencing genes. Materials and methods: Gene expression was studied in K562 cells previously exposed for 24 hours to two concentrations of quercetin (0.2 and 20 mM) dissolved in DMSO and 25 μg/ml hydroxyurea (HU) as a positive control. The exposed cells and controls were collected, and cell viability and proliferation parameters were evaluated microscopically. Variation in gene expression after CPMLE exposition was quantified from the total RNA isolated from cultured cells, using quantitative Real Time PCR. The studied genes were α, β and γ-globin genes, as well as the HbF regulators genes MYB, KLF1, BCL11A and BGLT3, and GAPDH as reference. Results: The proliferation rates were calculated as the ratio between the value at 24h and the initial number of cells (1X105 cells/well). The results for the quercetin concentrations of 0,2 and 20 mM were of 1,95 and 0,967, respectively, while for vehicle (DMSO) the value was 2,40. The percentages obtained for the viability, as assessed by trypan blue staining, were of 95,18% in vehicle, 91,40% and 88.99% for the quercetin concentrations of 0,2 and 20 mM, respectively. Altogether, these results indicate that quercetin slightly affects the proliferation and viability of the K562 cell line at both concentrations, although without cytotoxic effects. Transcriptional analysis demonstrated that both concentrations inhibit BCL11A, MYB, KLF1 and HBB gene expression levels; and increase the expression of HBG and BGLT3. The effect of quercetin on BCL11A gene expression was similar to the effect of HU, however the expression of MYB, KLF1, HBB and BGLT3 genes differed between the two molecules. In addition, the 0.2 mM concentration induced an overexpression of the HBA gene, similar to the effect of HU. Conclusion: The results presented are preliminary, however it is possible to observe that quercetin can modulate the expression of HbF, thus potentially constituting an effective alternative treatment of SCD. Acknowledgments This project was supported by Instituto Politécnico de Lisboa under the grant IDI&CA-IPL/2021/EpiCa/ESTeSL and partially supported by FCT/MCTES (UIDB/05608/2020 and UIDP/05608/2020). The authors are grateful to Fernando Nunes for kindly providing the Carica Papaya leaves. The authors do not declare any conflict of interest